Various solid samples can be ionized by matrix-assisted laser desorption/ionization (MALDI). Usually, MALDI is used as MALDI-TOF by combining with time-of-flight (TOF) mass spectrometer. Since a MALDI-TOF mass spectrometer (MS) is sensitive, widely applicable, and rapid to analyze samples, it is extensively used to analyze molecular structures of various solid substances, especially biological molecules.
However, since the reproducibility of MALDI mass spectra is very poor, it is difficult to utilize MALDI mass spectrometry for quantitative analysis of an analyte. For this reason, the industrial and scientific applicability of MALDI mass spectrometry is very limited.
Notwithstanding, various methods utilizing MALDI mass spectra, such as a relative quantitative analysis without using an internal standard, an absolute quantitative analysis using an internal standard, an absolute quantitative analysis by standard addition, etc., have been developed in order for quantitative analysis using MALDI mass spectra.
A relative quantitative analysis without an internal standard (or profile analysis) is a MALDI mass spectrometry that utilizes a classification algorithm, based on the fact that the relative signal intensity of each component in MALDI spectrum is constant, in order to analyze reproducibly a MALDI mass spectrum. However, the profile analysis has drawbacks that the design and performance of experiments are difficult.
In addition, a relative quantitative analysis with an internal standard is MALDI mass spectrometry that quantifies analytes by measuring the relative ratio of the peak height or area of each analyte to that of the internal standard from MALDI spectra of a sample containing the internal standard. However, the absolute amount of the analytes cannot be measured by the relative quantitative analysis with an internal standard.
Furthermore, an absolute quantitative analysis is MALDI mass spectrometry that obtains the absolute amount of an analyte by determining a calibration curve from a plurality of samples containing an internal standard with changing the amount of the analyte and, then, substituting the calibration curve with the relative quantity of the analyte obtained by a relative quantitative analysis with an internal standard. However, the absolute quantitative analysis has a drawback to obtain a calibration curve for each analyte for analyzing a sample containing a plurality of analytes.
Moreover, an absolute quantitative analysis by standard addition is MALDI mass spectrometry that determines the absolute amount of an analyte by using calibration points obtained from MALDI spectra of each sample which is prepared by dividing each unknown sample into two or more portions and adding known amount(s) of the analyte to these portions. However, the absolute quantitative analysis by standard addition has drawbacks to prepare an additional analyte to be analyzed, and many samples in order to analyze one analyte.
In order for quantitative analysis using MALDI spectra by the conventional methods, an internal standard, especially an isotopically labeled analyte, is used. However, it is very expensive to isotopically label the analyte such as high molecular weight material, for example, proteins, nucleic acids, etc., as well as low molecular weight material, for example, peptides. In addition, it is one of the drawbacks of quantitative MALDI mass spectrometry with an internal standard that pre-treatment of the analyte is not simple.
Since a MALDI sample is generally a mixture of an analyte and a matrix, MALDI spectra exhibit an analyte ion (AH+) and its fragmented products, and a matrix ion (MH+) and its fragmented products. Thus, MALDI spectral patterns are determined by the fragmentation patterns of AH+ and MH+ and the abundance (intensity) ratio of AH+ and MH+.
Ions generated by MALDI may decay inside the ion source (in-source decay, ISD) or outside the ion source (post-source decay, PSD). The reaction rate of ISD is fast and, thus, ISD terminates early. In contrast, the reaction rate of PSD is slow. These reaction rate and yield of fragmentation of the ions is determined by the reaction rate constant and the internal energy of the ions. Therefore, if the effective temperature of the plume generated by laser pulse in MALDI is found, the internal energy can be estimated and the reaction rate can be obtained by using the internal energy.
Many scientific researches to find out the temperature of the plume which includes ions generated by laser irradiation on MALDI samples and neutral molecules, have been carried out (J. Phys. Chem. 1994, 98, 1904-1909; J. Am. Soc. Mass Spectrum. 2007, 18, 607-616; J. Phys. Chem. A 2004, 108, 2405-2410).
However, the present inventors presented for the first time the best systematic method for measuring the plume temperature (J. Phys. Chem. B 2009, 108, 2405-2410). The present inventors succeeded in obtaining the reaction rate of ion fragmentation and the effective temperature by kinetic analysis of the time-resolved photodissociation spectra and the PSD spectra. In addition, the present inventors found out that the thus obtained temperature is the late plume temperature (Tlate). The present inventors determined the early plume temperature (Tearly) by analyzing ISD yields using the thus obtained reaction rate function.
Firstly, fragmented ion products abundance for ISD and PSD of peptide ions in MALDI mass spectra was measured. From these data, the survival probabilities of the peptide ion at the ion source exit (Sin) were evaluated. In consideration of experimental conditions, the maximum reaction constant at the ion source exit was obtained and, then, the maximum internal energy of the peptide ion was obtained from this maximum reaction constant. Varying the temperature, the internal energy distribution of the peptide ion was obtained and Tearly was determined to be the same temperature at which the probability of the region smaller than the maximum internal energy is equal to Sin.
The early and late temperatures of the ion-containing gas (plume), determined by the present inventors, were similar to those reported by other researchers. However, the method by the present inventors is much more systematic than the methods by other researchers and, thus, may be universally applicable (Journal of The American Society for Mass Spectrometry, 2011, vol. 22, pp 1070-1078). The disclosure of this prior document is incorporated herein by reference in its entirety.
Through these researches, the present inventors discovered, surprisingly, that, although the early plume temperature (Tearly) changes with change of MALDI experimental conditions, the fragmentation patterns of each ion are the same, respectively, when observing the mass spectra where Tearly is the same, out of the spectra obtained at various experimental conditions.
Surprisingly, the present inventors also discovered that, although the temperature (Tearly) at which ions are generated changes with change of the reaction conditions of the ion generation in MALDI, the total ion count (TIC) of each spectrum is the same, respectively, when observing the mass spectra where Tearly is the same, out of the spectra obtained at various experimental conditions.
Moreover, from the fact that the pattern of a mass spectrum as well as the total ion count (TIC) are the same when Tearly is the same, the present inventors further discovered that mass spectra at the same Tearly can be obtained when Tearly is kept constant by adjusting the laser pulse energy irradiated on a sample.
Accordingly, the present inventors have discovered that quantitative analysis by a mass spectrometer is possible since mass spectra of the same Tearly can be selected by utilizing the factors for measuring Tearly, such as the ion fragmentation pattern and the total ion count in MALDI spectra.
Furthermore, the present inventors have discovered that the reaction quotient (Q=[M][AH+]/([MH+][A])) of the proton exchange reaction in plume obtained from MALDI spectra with the same Tearly remains constant regardless of the analyte concentration in a sample.
That is, the present inventors have understood that, in MALDI-TOF mass spectrometry, early plume is nearly in an equilibrium state and the reaction quotient (Q) corresponds to the reaction constant (K) of the proton exchange reaction between the matrix and the analyte. Therefore, the present inventors have noted that the analyte-to-matrix ion intensity of MALDI-TOF mass spectra obtained at a certain temperature is proportional to the analyte-to-matrix mole ratio in a solid sample and, thereby, quantitative analysis can be performed.
The present inventors have invented a method for measuring an equilibrium constant of a proton transfer reaction between a matrix and an analyte by measuring MALDI mass spectra with change of MALDI ionization reaction conditions, comparing fragmentation patterns of matrix ions, analyte ions or other additive's ions contained in MALDI samples, selecting spectra of which fragmentation patterns of these materials are the same, and measuring the ratio of the matrix ion signal intensity to the analyte ion signal intensity from the selected MALDI spectra.
Also, the present inventors have invented a method for obtaining a calibration curve according to change of concentration ratio of the matrix to the analyte at a certain temperature by utilizing the reaction constant between the matrix and the analyte.
In addition, the present inventors have invented a method for quantitative analysis of an analyte in a sample from the moles of the analyte obtained by substituting the calibration curve with the matrix concentration and the ratio of the matrix ion signal intensity to the analyte ion signal intensity measured from MALDI mass spectra of the sample prepared by mixing the known amount of the matrix and the unknown amount of the anlyte.
Furthermore, the present inventors have invented a method for improving accuracy of quantitative mass spectrometry by suppressing the matrix signal suppression effect through dilution of the anlyte when the matrix signal suppression effect is more than 70%, in order to solve the problem that makes an accurate quantitative analysis difficult due to decrease of the matrix ion signal intensity and other analyte ion signal intensity when the concentration of the anlyte in the sample is very high.